This revised proposal is submitted in response to PA-04-119 Biodefense and Emerging Infectious Disease Research Opportunities. The experiments proposed address the mechanism(s) by which Ebola viruses (EBOV) evade the host cell interferon (IFN)alpha/beta response, a critical component of host innate immunity to viruses. EBOV are NIAID category A pathogens which cause highly lethal viral hemorrhagic fever. This proposal builds on previous studies performed by the PI and his collaborators which demonstrated that the Ebola virus VP35 protein acts as an "IFN-antagonist;" that is, VP35 is able to prevent in cells establishment of an IFNalpha/beta-induced antiviral state by blocking a critical step in the induction of the host IFN response, the activation of interferon regulatory factor 3 (IRF-3), (Basler et al. PNAS 97: 12289-12294, 2000;Easier et al. J. Virol. 77:7945-56, 2003). A recent study finds that VP35 blocks multiple IFNalpha/beta-inducing pathways including a pathway activated by RIG-I, a putative RNA helicase which is reportedly critical for the virus and dsRNA-induced production of IFNalpha/beta. This proposal expands on these earlier studies and seeks to define the mechanism(s) by which VP35 blocks signaling pathways that lead to the production of IFNalpha/beta and to determine the importance of this "IFN-antagonist" function for Ebola virus replication. The specific aims of the proposal are to: 1. Analyze the mechanism by which VP35 inhibits signaling to IRF-3 by the cellular RNA helicase RIG-I. 2. Define the specific IRF-3 phosphorylation events inhibited by VP35 and assess the impact of VP35 on the IRF3 kinases IKKepsilon and TBK-1. 3. Determine how participation by VP35, a multifunctional protein required for viral RNA synthesis and assembly, in viral polymerase and nucleocapsid complexes affects its "interferon-antagonist" function and define regions of VP35 required for interferon-antagonist function. 4. Generate VP35 mutant Ebola viruses and characterize their ability to antagonize the host IFN-response. This last aim will seek to correlate the mechanistic data obtained in Aims 1 and 2 with observations made in EBOV-infected cells and determine the importance of VP35 "IFN-antagonist" activity for virus replication by generating and characterizing mutant, recombinant EBOV.